Cryopreservation of winter-dormant mulberry buds using two-step freezing

Genetic resources of mulberry trees are commonly preserved as trophosomes, which are vulnerable to environmental factors, such as natural disasters, diseases, and pests. This study establishes a basic protocol for ultra-low temperature cryopreservation of mulberry trees using a two-step freezing process. The procedure was established using the ‘‘Daeshim’’ variety and then tested on genetic resources from 24 other mulberry varieties. Samples were first dried to a moisture content of 33–43% in a low-temperature forced-air chamber at −5 °C, then slowly frozen from −5 °C to −20 °C, and preserved in liquid nitrogen (−196 °C). To determine the regeneration rate, isolated dormant buds were inoculated into MS basal medium, and grown shoots were grafted onto 1-year-old rootstock via chip budding and then cultured. After freezing in liquid nitrogen, the ‘‘Daeshim’’ variety exhibited a survival and regeneration rate of more than 70% and 50%, respectively. Applying the two-step freezing process to genetic resources from 24 mulberry species yielded average survival and regeneration rates of 85.3% and 75.5%, respectively. Morus alba showed survival and regeneration rates of 100%, confirming the efficacy of the two-step freezing method. These results indicate the high feasibility of ultra-low-temperature cryopreservation through two-step freezing of dormant buds from mulberry genetic resources. Additional research is required into the variations in regeneration rates with freezing period in liquid nitrogen. KCI Citation Count: 0